drugs of abuse, neurological reviews and protocols

Humana Press
Drugs of
Abuse
Neurological Reviews
and Protocols
Edited by
John Q. Wang
Humana Press
M E T H O D S I N M O L E C U L A R M E D I C I N E
TM
Drugs of
Abuse
Neurological Reviews
and Protocols
Edited by
John Q. Wang
Gene Expression in Addiction 3
3
From: Methods in Molecular Medicine, vol. 79: Drugs of Abuse: Neurological Reviews and Protocols
Edited by: J. Q. Wang © Humana Press Inc., Totowa, NJ
1
The Temporal Sequence of Changes
in Gene Expression by Drugs of Abuse
Peter W. Kalivas, Shigenobu Toda, M. Scott Bowers,
David A. Baker, and M. Behnam Ghasemzadeh
1. Introduction
Addiction is a complex maladaptive behavior produced by repeated exposure
to rewarding stimuli (1). There are two primary features of addiction to all
forms of natural and pharmacological stimuli. First, the rewarding stimulus
associated with the addiction is a compelling motivator of behavior at the
expense of behaviors leading to the acquisition of other rewarding stimuli.

Thus, individuals come to orient increasing amounts of their daily activity
around acquisition of the rewarding stimulus to which they are addicted.
Second, there is a persistence of craving for the addictive stimulus, combined
with an inability to regulate the behaviors associated with obtaining that
stimulus. Thus, years after the last exposure to an addictive stimulus, reexposure
to that stimulus or environmental cues associated with that stimulus will elicit
behavior seeking to obtain the reward.
During the course of repeated exposures to strong motivationally relevant
stimuli specifi c brain nuclei and circuits become engaged that mediate the
addicted behavioral response. It is generally thought that different rewarding
stimuli involve different brain circuits, but that regions of overlap with other
motivational stimuli exist, forming a common substrate for all addictive
stimuli. Studies using animal models of reward and addiction have focused
on subcortical brain circuits known to be involved in drug reward, such as the
dopamine projection from the ventral mesencephalon to the nucleus accumbens
(2,3). Accordingly, molecular and electrophysiological studies of the cellular
plasticity mediating the emergence of addictive behaviors have focused on
4 Kalivas et al.
the nucleus accumbens and ventral mesencephalon. However, about 5 yr ago
studies emerged from both the animal literature and neuroimaging of drug
addicts indicating that the expression of addicted behaviors such as sensitiza-
tion and craving involved regions of the cortex and allocortex (4–7). In this
regard, two regions that have come to be closely associated with addiction are
the amygdala and frontal cortex (including the anterior cingulate and ventral
orbitofrontal cortex). In addition, the last decade of research has revealed a
variety of enduring changes in gene expression produced by repeated exposure
to drugs of abuse, notably psychostimulants (3,8). The most long-lasting
neuroadaptations that would be expected to underlie enduring behaviors
associated with addiction appear to be concentrated in the nucleus accumbens
and in cortical regions providing input to the nucleus accumbens, such as

the prefrontal cortex. These studies are outlined and integrated with the
corticostriatal circuitry postulated to be critical for the expression of behavioral
characteristics of psychostimulant addiction, such as sensitization and craving.
2. Temporal and Anatomical Sequence of Changes
in Gene Expression
A variety of studies using different addictive drugs, given in different
dosing regimens and employing different withdrawal periods, have shown that
repeated administration of addictive drugs produced short, intermediate, and
enduring changes in gene expression. Figure 1 illustrates the sequence of
changes in gene expression associated with repeated cocaine administration.
Five categories of cocaine-induced changes in gene expression are outlined,
ranging from increases in immediate early gene (IEG) expression that diminish
with repeated injections to changes in gene expression that appear only after
a period of withdrawal. The data outlined in Fig. 1 are specifi c for cocaine-
induced changes in gene expression, and using these data as a guide certain
temporal patterns of drug-induced changes in gene expression can be discerned
from the extant literature. Similarly, anatomical patterns of gene expression
related to various times during the chronic injection and withdrawal periods
can be shown. However, there are exceptions in the anatomical discretion, and,
importantly, in many brain nuclei relevant to addiction, notably the amygdala,
very little data have been collected regarding changes in gene expression.
3. Rapid Response and Tolerance, Widespread
in Dopamine Terminal Fields
The earliest changes in gene expression that are measurable shortly after
acute drug administration occur in many brain regions, the most well studied
being dopamine terminal fi elds such as the striatum, nucleus accumbens, and
prefrontal cortex. These genes include classic IEG transcription factors such
Gene Expression in Addiction 5
as c-fos and zif268 (9,10). However, both cytosolic IEGs such as homer1a
and arc and extracellular IEG-like proteins such as the pentraxin narp are

also induced in a number of brain regions by acute administration of cocaine
(11). The increase in these proteins is thought to initiate changes that partly
mediate the acute effects of drugs, as well as provide a background upon
Fig. 1. Temporal pattern of changes in gene expression produced by repeated
injections of cocaine.
6 Kalivas et al.
which subsequent, more enduring changes in gene expression can emerge. In
general, the proteins encoded by IEGs have a relatively short half-life, and
levels return to normal within 24 h after the injection. Moreover, with repeated
administration of drug the induction produced by each injection becomes
progressively less until by 1 wk of injection little or no induction is produced.
4. Slow Progressive Change and Rapid Return,
Predominately in the Ventral Tegmental Area
Another category of changes in gene expression are those that gradually
accumulate with repeated administration but disappear within a few days
after the last injection. Interestingly, many changes in gene expression in
this category are found in dopamine or nondopamine cells in the ventral
tegmental area (VTA). Included are proteins encoded by genes that are directly
related to dopamine transmission, such as tyrosine hydroxylase and dopamine
transporters (12–14). In addition, genes associated with dopamine receptor
signaling such as Giα undergo a short-term change in expression after the last
cocaine injection (15). Notably, the expression of genes related to glutamate
transmission such as GluR1 and NMDAR2 are also included in this category
(16,17). Taken together these changes in gene expression appear to facilitate
glutamatergic activation of cells in the VTA while simultaneously diminishing
the capacity of D2 dopamine autoreceptors to provide negative regulation of
dopamine cell fi ring (18,19). These changes probably contribute to known
physiological alterations in dopamine cell function associated with short-term
withdrawal such as increased dopamine cell fi ring and enhanced releasibility
of dopamine, glutamate, and γ-aminobutyric acid (GABA) in the VTA (20,23).

In addition, the disinhibition of dopamine cells may contribute to the increased
releasibility of dopamine in axon terminal fi elds such as the nucleus accumbens
and striatum.
5. Slow Change, Slow Return, Predominately
in the Striatal Dopamine Terminal Fields
This category of cocaine-induced changes in gene expression has recently
received considerable attention as possible mediators of the transition from
casual to addictive patterns of drug-taking (24). Some of these genes are
IEG-like in that they are induced by acute drug administration. However, the
proteins have a relatively long half-life. As a result elevated protein levels are
present for an extended period, as long as weeks after the last drug injection.
The classic gene in the category is ∆-fosB, which has been shown to accumulate
in the striatum with repeated psychostimulant exposure (25). Notably, the
increased expression is also associated with a redistribution of cellular expres-
sion into different striatal compartments (26). In addition, changes in gene
Gene Expression in Addiction 7
expression and protein function associated with D1 receptor signaling fall into
this category, including an induction in protein kinase A, mitogen-activated
protein (MAP) kinase, and phospho-cAMP response element binding protein
(phospho-CREB) (24). Accordingly, genes regulated by phospho-CREB,
such as preprodynorphin, are also altered by repeated cocaine administration
and endure for weeks after the last injection (27,28). Likewise, while gene
expression may not be altered, proteins regulated by protein kinase A (PKA),
CdK5, or MAP kinase phosphorylation demonstrate altered function for an
extended withdrawal period after the last drug injection, including sodium
channels and the cystine/glutamate antiporter in the striatum (29). In addition,
proteins related to glutamate transmission show the slow change/slow return
pattern of expression, including mGluR5, which has been recently linked
to cocaine reward (30,31). Also, proteins involved in other neurotransmitter
systems in the striatal complex, including histidine decarboxylase and the

adenosine transporter, show this temporal pattern (30,32,33). These changes
play a signifi cant role in some of the enduring changes in excitability in spiny
cells in the nucleus accumbens and striatum. Notably, spiny cells show more
avid inhibition in response to D1 receptor stimulation and have a decreased
postsynaptic response to α-amino-3-hydroxy-5-methyl-4-isoxazole propionic
acid (AMPA) receptor stimulation or long-term potentiation in response
to tetanic stimulation of glutamatergic afferents to the nucleus accumbens
(19,34).
6. Changes Only During Withdrawal, Enduring for Weeks,
Predominately in the Prefrontal Cortex and Nucleus Accumbens
Members of this category of genes have undergone recent intensive study
and the changes in expression generally appear after only a week or more
of withdrawal from repeated drug administration. The changes are almost
exclusively in the prefrontal cortex and nucleus accumbens and include a
variety of gene products involved in neurotransmission, cell signaling, and
glial function. However, the changes are notable in that they endure for weeks
and involve a predominance of genes affecting glutamate transmission relative
to dopamine transmission. Genes in this category altered by cocaine encode
mGluR1, mGluR2/3, homer1bc, GluR5, A1 adenosine receptor, TrkB, BDNF,
AGS3, Giα, GFAP, and vimentin. These changes in expression combine to
produce a generalized decrease in signaling through group I and group II
mGluR and in general serve to decrease excitability of cells in the nucleus
accumbens (30,35,36). In addition, the changes in glial fi brillary acidic protein
(GFAP) and vimentin suggest an enduring activation of glia, which may
contribute to the reduction in extracellular glutamate in the nucleus accumbens
that is associated with repeated cocaine administration (37).
8 Kalivas et al.
7. Rebounding IEG
This is a category that to date contains a single gene product, nac-1 (38,39).
This protein has expression characteristics of the IEG class in that levels are

induced by acute drug administration, and progressive tolerance to this induc-
tion occurs with repeated administration. However, similar to late expressing
genes, the levels of nac-1 rise at 1 wk of withdrawal and are maintained for
at least 3 wk thereafter. Experiments using viral overexpression of nac-1 and
antisense oligonucleotide inhibition of protein expression reveal that nac-1 is
important in the development of behavioral sensitization and in the acquisition
of cocaine self-administration.
8. Anatomical Sequence of Gene Expression
and the Development of Enduring Changes in Reward Circuitry
As outlined in the preceding, different brain regions demonstrate the major-
ity of changes in gene expression in a temporal sequence. Changes to acute
administration are very widespread, predominately in dopamine axon terminal
fi elds. A large number of alterations in gene expression that exist for a relatively
short duration after discontinuing repeated drug administration are found in
the VTA. These changes may contribute to an increased responsiveness of
dopamine cells to acute drug injection that will promote more enduring changes
in gene expression in dopamine axon terminal fi elds such as the prefrontal
cortex and nucleus accumbens. In the dopamine terminal fi elds the expression
of proteins undergoes a transition from those that are produced during repeated
drug administration and endure for a period of time after injection to changes
in expression that develop later in withdrawal and endure for an extended
period after the last drug injection. This temporal transition in gene expression
can be seen as constituting a new baseline of cellular functioning that mediates
the expression of behaviors associated with addiction, such as drug craving
and sensitization. Notably, these enduring changes in expression are in the
prefrontal cortex and nucleus accumbens, and the relationship between these
two regions has come under increasing scrutiny as the site of primary pathology
in psychostimulant addiction.
9. Conclusions
The studies reviewed in this chapter point to the possibility of a final

common pathway, and possibly similar cellular neuroadaptations between
drugs and stimuli that provoke craving and relapse. The extant data support
a role of the projection from the prefrontal cortex to nucleus accumbens in
the expression of addiction-related behaviors, such as sensitization and drug-
seeking behavior, and there is abundant evidence for enduring neuroadaptations
Gene Expression in Addiction 9
in gene expression and neuronal function in these brain regions following a
bout of drug-taking. Although the studies outlined in this chapter are promising
in pointing to a common point of intervention in addiction to various chemical
classes of drugs, it is important to note that such a generalization based
primarily on work with psychostimulants is premature, and verifi cation will
require substantially more research using other classes of drugs. Also, the
temporal sequence of neuroadaptive changes during drug withdrawal points
to the possible utility of targeting different pharmacotherapies at different
stages of withdrawal. Thus, in early withdrawal drugs affecting dopamine
transmission may be more effective, while in later withdrawal modulation of
glutamate transmission may be more effi cacious.
References
1. O’Brien, C. (2001) Drug addiction and drug abuse, in The Pharmacological Basis
of Therapeutics (Hardman, J., Limbird, L., and Gilman, A. G., eds.) McGraw-Hill,
New York, pp. 621–642.
2. Koshiya, K. and Kato, T. (1983) Acute changes in nigral substance P content
induced by drugs acting on dopamine, muscarine and GABA receptors. Naunyn
Schmiedebergs Arch. Pharmacol. 324, 223–227.
3. Unterwald, E. M., Cox, B. M., Kreek, M. J., Cote, T. E., and Izenwasser, S. (1993)
Chronic repeated cocaine administration alters basal and opioid-regulated adenylyl
cyclase activity. Synapse 15, 33–38.
4. Pierce, R. C. and Kalivas, P. W. (1997) A circuitry model of the expression of
behavioral sensitization to amphetamine-like psychostimulants. Brain Res. Rev.
25, 192–216.

5. Volkow, N. D. and Fowler, J. S. (2000) Addiction, a disease of compulsion and
drive: involvement of the orbitofrontal cortex. Cereb. Cortex 10, 318–325.
6. Grant, S., London, E. D., Newlin, D. B., et al. (1996) Activation of memory
circuits during cue-elicited cocaine craving. Proc. Natl. Acad. Sci. USA 93,
12040–12045.
7. Childress, A. R., Mozley, P. D., McElgin, W., Fitzgerald, J., Reivich, M., and
O’Brien, C. P. (1999) Limbic activation during cue-induced cocaine craving. Am.
J. Psychiatry 156, 11–18.
8. White, F. J. and Kalivas, P. W. (1998) Neuroadaptations involved in amphetamine
and cocaine addiction. Drug Alcohol Depend. 51, 141–154.
9. Daunais, J. and McGinty, J. F. (1996) The effects of D1 or D2 dopamine receptor
blockade on zif /268 and preprodynorphin gene expression in rat forebrain follow-
ing a short-term cocaine binge. Mol. Brain Res. 354, 237–248.
10. Moratalla, R., Vickers, E. A., Robertson, H. A., Cochran, B. H., and Graybiel, A. M.
(1993) Coordinate expression of c-fos and jun B is induced in the rat striatum by
cocaine. J. Neurosci. 13, 423–433.
11. Lanahan, A. and Worley, P. (1998) Immediate-early genes and synaptic function.
Neurobiol. Learn. Mem. 70, 37–43.
10 Kalivas et al.
12. Vrana, S. L., Vrana, K. E., Koves, T. R., Smith, J. E., and Dworkin, S. I. (1993)
Chronic cocaine administration increases CNS tyrosine hydroxylase enzyme
activity and mRNA levels and tryptophan hydroxylase enzyme activity levels.
J. Neurochem. 61, 2262–2268.
13. Sorg, B. A., Chen, S. Y., and Kalivas, P. W. (1993) Time course of tyrosine
hydroxylase expression following behavioral sensitization to cocaine. J. Pharma-
col. Exp. Ther. 266, 424–430.
14. Xia, Y., Goebel, D. J., Kapatos, G., and Bannon, M. J. (1992) Quantitation of
rat dopamine transporter mRNA: effects of cocaine treatment and withdrawal.
J. Neurochem. 59, 1179–1182.
15. Nestler, E. J., Terwilliger, R. Z., Walker, J. R., Sevarino, K. A., and Duman, R. S.

(1990) Chronic cocaine treatment decreases levels of the G protein subunits G
iα

and G
oα
in discrete regions of rat brain. J. Neurochem. 55, 1079–1082.
16. Fitzgerald, L. W., Ortiz, J., Hamedani, A. G., and Nestler, E. J. (1996) Drugs
of abuse and stress increase the expression of GluR1 and NMDAR1 glutamate
receptor subunits in the rat ventral tegmental area: common adaptions among
cross-sensitizing agents. J. Neurosci. 16, 274–282.
17. Churchill, L., Swanson, C. J., Urbina, M., and Kalivas, P. W. (1999) Repeated
cocaine alters glutamate receptor subunit levels in the nucleus accumbens and
ventral tegmental area of rats that develop behavioral sensitization. J. Neurochem.
72, 2397–2403.
18. Wolf, M. E., White, F. J., Nassar, R., Brooderson, R. J., and Khansa, M. R.
(1993) Differential development of autoreceptor subsensitivity and enhanced
dopamine release during amphetamine sensitization. J. Pharmacol. Exp. Ther.
264, 249–255.
19. White, F. J., Hu, X. T., Zhang, X. F., and Wolf, M. E. (1995) Repeated administra-
tion of cocaine or amphetamine alters neuronal responses to glutamate in the
mesoaccumbens dopamine system. J. Pharmacol. Exp. Ther. 273, 445–454.
20. White, F. J., et al. (1995) Neurophysiological alterations in the mesocorticolimbic
dopamine system during repeated cocaine administration., in The Neurobiology of
Cocaine Addiction (Hammer, R., ed.), CRC Press, Boca Raton, FL, pp. 99–120.
21. Kalivas, P. W. and Duffy, P. (1993) Time course of extracellular dopamine and
behavioral sensitization to cocaine. II. Dopamine perikarya. J. Neurosci. 13,
276–284.
22. Kalivas, P. W. and Duffy, P. (1998) Repeated cocaine administration alters extracel-
lular glutamate in the ventral tegmental area. J. Neurochem. 70, 1497–1502.
23. Bonci, A. and Williams, J. T. (1996) A common mechanism mediates long-term

changes in synaptic transmission after chronic cocaine and morphine. Neuron
16, 631–639.
24. Nestler, E. (2001) Molecular basis of long-term plasticity underlying addiction.
Nat. Rev. 2, 119–128.
25. Nestler, E. J., Barrot, M., and Self, D. W. (2001) DeltaFosB: a sustained molecular
switch for addiction. Proc. Natl. Acad. Sci. USA 98, 11042–11046.
Gene Expression in Addiction 11
26. Moratalla, R., Elibol, B., Vallejo, M., and Graybiel, A. M. (1996) Network-level
changes in expression of inducible fos-jun proteins in the striatum during chronic
cocaine treatment and withdrawal. Neuron 17, 147–156.
27. Hurd, Y. L. and Herkenham, M. (1993) Molecular alterations in the neostriatum
of human cocaine addicts. Synapse 13, 357–369.
28. Kelz, M. B, Chen, J., Carlezon, W. A., Jr., et al. (1999) Expression of the transcrip-
tion factor deltaFosB in the brain controls sensitivity to cocaine. Nature 401,
272–276.
29. Zhang, X. F., Hu, X. T., and White, F. J. (1998) Whole-cell plasticity in cocaine
withdrawal: reduced sodium current in nucleus accumbens neurons. J. Neurosci.
18, 488–498.
30. Swanson, C. J., Baker, D. A., Carson, D., Worley, P. F., and Kalivas, P. W. (2001)
Repeated cocaine administration attenuates group I metabotropic glutamate
receptor-mediated glutamate release and behavioral activation: a potential role for
Homer 1b/c. J. Neurosci. 21, 9043–9052.
31. Chiamulera, C., Epping-Jordan, M. P., Zocchi, A., et al. (2001) Reinforcing and
locomotor stimulant effects of cocaine are absent in mGluR5 null mutant mice.
Nat. Neurosci. 4, 873–874.
32. Kubota, Y., Ito, C., Kuramasu, A., Sato, M., and Watanabe, T. (1999) Transient
increases of histamine H1 and H2 receptor mRNA levels in the rat striatum after
the chronic administration of methamphetamine. Neurosci. Lett. 275, 37–40.
33. Ito, C., Onodera, K., Sakurai, E., Sato, M., and Watanabe, T. (1997) Effect of
cocaine on the histaminergic neuron system in the rat brain. J. Neurochem. 69,

875–878.
34. Henry, D. J. and White, F. J. (1995) The persistence of behavioral sensitization to
cocaine parallels enhanced inhibition of nucleus accumbens neurons. J. Neurosci.
15, 6287–6299.
35. Manzoni, O., Pujalte, D., Williams, J., and Bockaert, J. (1998) Decreased pre-
synaptic sensitivity to adenosine after cocaine withdrawal. J. Neurosci. 18,
7996–8002.
36. Pierce, R., Pierce-Bancroft, A., and Prasad, B. (2000) Neurotrophin-mediated
second messengers contribute to the initiation of behavioral sensitization to
cocaine. J. Neurosci. 20, 1–34.
37. Haile, C. N., Hiroi, N., Nestler, E. J., and Kosten, T. A. (2001) Differential
behavioral responses to cocaine are associated with dynamics of mesolimbic
dopamine proteins in Lewis and Fischer 344 rats. Synapse 41, 179–190.
38. Cha, X. Y., Pierce, R. C., Kalivas, P. W., and Mackler, S. A. (1997) NAC-1, a rat
brain mRNA, is increased in the nucleus accumbens three weeks after chronic
cocaine self-administration. J. Neurosci. 17, 6864–6871.
39. Mackler, S. A., Korutla, L., Cha, X. Y., Koebbe, M. J., Fournier, K. M., Bowers,
M.S., and Kalivas, P. (2000) NAC-1 is a brain POZ/BTB protein that can prevent
cocaine-induced sensitization in the rat. J. Neurosci. 20, 6210–6217.
12 Kalivas et al.
Psychostimulants and Glutamate Receptors 13
13
From: Methods in Molecular Medicine, vol. 79: Drugs of Abuse: Neurological Reviews and Protocols
Edited by: J. Q. Wang © Humana Press Inc., Totowa, NJ
2
Effects of Psychomotor Stimulants
on Glutamate Receptor Expression
Marina E. Wolf
1. Introduction: Addiction as a Form
of Glutamate-Dependent Plasticity

It is increasingly well accepted that addiction can be viewed as a form
of neuronal plasticity, even as a type of very powerful, albeit maladaptive,
learning. On a behavioral level, this can be conceptualized as the transition from
experimentation to compulsive drug-seeking behavior. This view of addiction
has been strengthened by many recent studies demonstrating commonalities
between mechanisms underlying learning and addiction. Both are associated
with changes in gene expression, phosphorylation and phosphatase cascades,
neurotrophin signaling, altered dendritic morphology, and activity-dependent
forms of plasticity such as long-term potentiation (LTP) and long-term depres-
sion (LTD) (1,2). Through these mechanisms, drugs of abuse are proposed to
strengthen or weaken activity in pathways related to motivation and reward.
This in turn may produce behavioral changes that drive compulsive drug-
seeking behavior in addiction, including sensitization of incentive-motivational
effects of drugs, enhanced ability of drug-conditioned stimuli to control
behavior, and loss of inhibitory control mechanisms that normally govern
reward-seeking behavior (3,4).
An open question is how drugs of abuse, which initially target monoamine
receptors, are able to infl uence mechanisms of synaptic plasticity. Glutamate
is a key transmitter for synaptic plasticity, and many neuronal pathways
implicated in addiction are glutamatergic (4). Historically, studies of behavioral
sensitization, a well-established animal model for addiction, were important in
directing drug addiction research toward glutamate (5). Behavioral sensitization
14 Wolf
refers to the progressive enhancement of species-specifi c behavioral responses
that occurs during repeated drug administration and persists even after long
periods of withdrawal. Although most studies have measured sensitization
of locomotor activity, sensitization also occurs to the reinforcing effects of
psychomotor stimulants. Behavioral sensitization is infl uenced by the same
factors that infl uence addiction (stress, conditioning, and drug priming), and is
accompanied by profound cellular and molecular adaptations in the neuronal

circuits that are fundamentally involved in normal motivated behavior as well
as addiction. Like addiction, it is extremely persistent. Robinson and Berridge
(6,7) have argued for an incentive-sensitization view of addiction, which holds
that repeated drug administration sensitizes the neuronal systems involved in
drug “wanting” rather than drug “liking.”
It is now acknowledged that the development of sensitization requires
glutamate transmission in the midbrain, where dopamine (DA) cell bodies
are located, whereas its maintenance and expression are associated with
profound changes in glutamate transmission in limbic and cortical brain regions
that receive dopaminergic innervation. To understand the role of glutamate
transmission in sensitization, many studies have examined drug effects on
glutamate transmission in these brain regions. This review focuses on cocaine
and amphetamine effects on glutamate receptor expression in the midbrain
(ventral tegmental area [VTA] and substantia nigra), the striatal complex
(nucleus accumbens [NAc] and dorsal striatum), and the prefrontal cortex
(PFC). Recent studies are emphasized, with the goal of updating a comprehen-
sive review published 4 yr ago (8).
2. Effects of Psychomotor Stimulants on Glutamate Receptor
Expression in the VTA and Substantia Nigra
2.1. Role of the VTA in Behavioral Sensitization
Many lines of evidence have suggested that the development of behavioral
sensitization is associated with an increase in excitatory drive to VTA DA
neurons (8). This provided the impetus for examining whether glutamate
transmission is enhanced in the VTA during the early phase of drug withdrawal.
The fi rst evidence to support this hypothesis came from in vivo single-unit
recording studies demonstrating that VTA DA neurons recorded from cocaine-
or amphetamine-sensitized animals were more responsive to the excitatory
effects of iontophoretic glutamate (9). A subsequent study showed that increased
responsiveness was selective for α-amino-3-hydroxy-5-methylisoxazole-
4-propionic acid (AMPA) (there was no change in sensitivity to N-methyl-

D-aspartate [NMDA] or a metabotropic glutamate receptor agonist) and
transient, present 3 but not 10–14 d after discontinuing repeated drug adminis-
Psychostimulants and Glutamate Receptors 15
tration (10). Recently, we have shown that AMPA receptor supersensitivity can
also be demonstrated in microdialysis experiments, by monitoring the ability
of intra-VTA AMPA to activate VTA DA neurons and thus increase DA levels
in the ipsilateral NAc. Using dual-probe microdialysis, we found that intra-
VTA administration of a low dose of AMPA produced signifi cantly greater DA
effl ux in the NAc of amphetamine-treated rats (11). This augmented response
was transient (present 3 but not 10–14 d after the last injection) and specifi c for
AMPA, as intra-VTA NMDA administration produced a trend toward increased
NAc DA levels that did not differ between groups. Thus, both microdialysis
and in vivo electrophysiological data suggest an enhancement of AMPA
receptor transmission onto VTA DA neurons during the early phase of drug
withdrawal. An increase in glutamate receptor expression would provide a
simple explanation for such fi ndings. For this and other reasons, a number of
studies have examined the effect of repeated drug administration on glutamate
receptor expression in VTA. Studies on glutamate receptor expression in the
substantia nigra are also considered. Although the substantia nigra has received
less attention in recent years than the VTA, it exhibits similar drug-induced
adaptations and is also implicated in the development of sensitization (8).
2.2. Results in the VTA and Substantia Nigra
Using Western blotting, Nestler and colleagues found increased GluR1
levels in the VTA of rats killed 16–18 h after discontinuation of repeated
cocaine, morphine, ethanol, or stress paradigms (12,13). Increased GluR1
was not observed in the substantia nigra after repeated cocaine or morphine
treatment (12). The substantia nigra was not examined in stress studies (12),
but after repeated ethanol administration, there was a greater increase in GluR1
in the substantia nigra than in the VTA (13). Repeated cocaine also increased
NR1 in VTA but had no effect on GluR2, NR2A/B, or GluR6/7 (12). Churchill

et al. (14) treated rats with saline or cocaine for 7 d (15 mg/kg on d 1 and
7, 30 mg/kg on d 2–6), measuring locomotor activity after the fi rst and last
injections; those rats that showed >20% increase in locomotor activity were
defi ned as sensitized. Then, protein levels of glutamate receptor subunits
were determined by Western blotting 24 h or 3 wk after daily injections were
discontinued. In agreement with results of Fitzgerald et al. (12), Churchill et al.
(14) found increased GluR1 and NR1 levels in the VTA of rats killed 1 d but not
3 wk after discontinuation of this different cocaine regimen. Interestingly, this
was observed only in those cocaine-treated rats that developed sensitization.
GluR2/3 was not measured after 1 d but was unaltered after 3 wk (14).
In contrast, our own quantitative immunoautoradiography studies found no
change in GluR1 immunoreactivity in VTA, substantia nigra, or a transitional
16 Wolf
area after 16–24 h of withdrawal from repeated amphetamine or cocaine
treatment (15). Importantly, this study (15) also failed to fi nd a change in GluR1
immunoreactivity after 3 or 14 d of withdrawal from the same amphetamine
regimen that resulted in enhanced electrophysiological (10) and neurochemical
(11) responsiveness to intra-VTA AMPA at the 3-d withdrawal time. Thus,
although part of the discrepancy between results from different labs may be
attributable to different drug regimens, our fi ndings suggest that increased
GluR1 expression is unlikely to explain our electrophysiological or neurochemi-
cal fi ndings of increased responsiveness to AMPA. Other possible reasons for
differences between our immunoautoradiography studies and prior Western
blotting studies have been discussed previously (15).
Another fi nding relevant to this controversy is that overexpression of GluR1
in the rostral VTA using a herpes simplex virus resulted in intensifi cation of the
locomotor stimulant and rewarding properties of morphine (16,17). Although
this is an interesting fi nding, it does not necessarily imply that increased
GluR1 expression is involved in the naturally occurring pathways that produce
behavioral sensitization to morphine or psychomotor stimulants. A state

resembling behavioral sensitization can be produced by a number of diverse
experimental manipulations, all sharing the ability to produce brief but intense
activation of VTA DA cells. These include repeated electrical stimulation of
the VTA (18) or PFC (19), and pharmacological disinhibition of VTA DA
cells (20).
In contrast to discrepant results at the protein level, all studies agree that
mRNA levels for AMPA receptor subunits in the VTA are not altered during
withdrawal from repeated amphetamine or cocaine. We found no change in
GluR1 mRNA using reverse transcriptase-polymerase chain reaction (RT-PCR)
in the VTA of rats killed 16–18 h after discontinuing repeated amphetamine or
cocaine administration (15). Similarly, Bardo et al. (21) used RNase protection
assays to quantify GluR1-4 mRNA levels in the ventral mesencephalon of
rats killed 30 min after the third or tenth amphetamine injection in a repeated
regimen and observed no signifi cant changes, although behavioral sensitization
was demonstrated. Ghasemzadeh et al. (22) used RT-PCR to determine mRNA
levels for GluR1-4, NR1, and mGluR5 in the VTA 3 wk after discontinuing
repeated cocaine or saline injections, and found no signifi cant changes as
a result of repeated cocaine treatment, although acute cocaine challenge
produced a small reduction in NR1 mRNA levels in the VTA of both naïve
and sensitized rats.
As noted previously, Western blotting studies have found increased NR1
levels in the VTA of rats killed 16–24 h (but not 3 wk) after discontinuing
repeated cocaine administration, suggesting that the increase is transient
(12,14). In contrast, using immunohistochemical methods, Loftis and Janowsky
Psychostimulants and Glutamate Receptors 17
(23) compared rats treated with repeated cocaine or saline and found signifi cant
increases in NR1 immunoreactivity in the cocaine group after 3 and 14 d
of withdrawal and a trend toward an increase after 24 h. Using the same
regimen as Churchill et al. (14), Ghasemzadeh et al. (22) found no signifi cant
changes in NR1 mRNA levels in VTA using RT-PCR. We used quantitative

immunoautoradiography to examine NR1 expression in VTA, substantia
nigra, and a transitional area in rats killed 3 or 14 d after discontinuing
repeated amphetamine administration. No changes were observed after 3 d
of withdrawal, whereas NR1 immunolabeling was signifi cantly decreased in
the intermediate and caudal portions of the substantia nigra, but not in other
midbrain regions, after 14 d of withdrawal (24). NR1 levels in the NAc and
prefrontal cortex were also decreased at this withdrawal time (24). It may
be relevant to note that although NMDA receptor transmission in the VTA
is required for the induction of sensitization, repeated stimulation of NMDA
receptors in the VTA is not suffi cient to elicit sensitization (25,26).
2.3. Summary: VTA and Substantia Nigra
As reviewed in Subheading 2.1., both neurochemical and electrophysi-
ological studies suggest that there is an enhancement in the responsiveness of
VTA DA neurons to the excitatory effects of AMPA shortly after discontinuing
repeated psychostimulant administration. An increase in AMPA receptor
expression in the VTA would provide a simple explanation for these results.
However, although Western blotting studies have found increased GluR1 and
NR1 levels in the VTA shortly after cocaine administration is discontinued,
this is not observed with immunoautoradiography following either cocaine
or amphetamine administration (see 15 for discussion). More importantly,
after the same drug regimens and withdrawal times that are associated with
increased responsiveness of VTA DA neurons to AMPA, no changes in GluR1
are observed. Thus, although considerable evidence suggests that enhanced
responsiveness of VTA DA neurons to AMPA is closely linked to the induc-
tion of sensitization, the mechanisms are likely to be more complex than a
generalized increase in GluR1 expression within the VTA.
As LTP is expressed as a potentiation of AMPA receptor transmission,
an alternative explanation is that sensitization is accompanied by LTP-like
changes that increase the effi ciency of glutamate transmission in the VTA.
Although LTP appears to involve insertion of AMPA receptor subunits into

synaptic sites (27), there is no evidence that this is accompanied by increases in
total cellular expression of AMPA receptor subunits. Supporting the involve-
ment of LTP in the development of sensitization, a single systemic injection of
cocaine to mice (suffi cient to elicit behavioral sensitization) produced LTP in
midbrain DA neurons (28). The mechanisms responsible are probably complex.
18 Wolf
DA-releasing stimulants could promote LTP by decreasing the opposing
infl uence of LTD, as D2 receptor activation inhibits LTD in midbrain slices
(29,30). Psychostimulant-induced increases in VTA glutamate levels may
also promote LTP (31,32). Of course, mechanisms unrelated to LTP may also
contribute to increased excitability of VTA DA neurons, including inhibition of
mGluR-mediated inhibitory postsynaptic potentials (IPSPs) (33,34). Finally, it
should be noted that glutamate transmission in the VTA may be infl uenced by
drug-induced alterations in other transmitter systems. Mechanisms that may
contribute to sensitization-related plasticity in the VTA have been reviewed
elsewhere (2,35).
An interesting future direction is to study sensitization in transgenic mice
with alterations in glutamate receptors or signaling pathways implicated in
LTP. Chiamulera et al. (36) reported that mGluR5 knockout mice do not exhibit
locomotor activation when injected with acute cocaine, and do not acquire
cocaine self-administration. Mao et al. (37) found that mGluR1 knockout mice
have augmented locomotor responses to amphetamine, perhaps due to impaired
mobilization of inhibitory dynorphin systems that normally regulate responses
to amphetamine. Using GluR1 knockout mice, Vekovischeva et al. (38) found
that sensitization was normal when mice received repeated morphine injections
in the same environment in which they were ultimately tested (context-dependent
sensitization) but did not develop when the repeated treatment was given in home
cages (context-independent sensitization), whereas wild-type mice developed
sensitization under both conditions. Although all of these results are potentially
important, it is hard to draw fi rm conclusions because of the possibility of

altered neuronal development in glutamate receptor defi cient mice.
3. Effect of Psychomotor Stimulants on Glutamate Receptor
Expression in the Nucleus Accumbens and Dorsal Striatum
3.1. Role of the NAc and Striatum in Behavioral Sensitization
The NAc occupies a key position in the neural circuitry of motivation
and reward. Not surprisingly, it is also critical for behavioral sensitization.
While psychostimulants act in the midbrain to trigger the development of
sensitization, drug actions in the NAc lead to the expression of a sensitized
response. Accordingly, the VTA is associated with transient cellular adaptations
during the early withdrawal period, while the NAc is the site of more persistent
adaptations (see refs. 10 and 39). The output neurons of the NAc, medium spiny
γ-aminobutyric acid (GABA) neurons, are regulated by convergent DA and
glutamate inputs, although the nature of the interaction between DA and gluta-
mate is complex and remains controversial (40). Repeated psychostimulant
administration leads to profound changes in both DA and glutamate trans-
Psychostimulants and Glutamate Receptors 19
mission in the NAc (8,39), and many recent studies have demonstrated
that glutamate transmission in the NAc plays a critical role in drug-seeking
behavior (4). Therefore, many groups have examined the effects of psycho-
stimulants on glutamate receptor expression in the NAc, as well as in the
dorsal striatum. The dorsal striatum exhibits many of the same drug-induced
adaptations as the NAc, although the NAc has received much more attention
in recent years (8).
3.2. Results in the NAc and Striatum
We have measured glutamate receptor subunit mRNA levels and immuno-
reactivity in rats treated for 5 d with 5 mg/kg of amphetamine or saline and
perfused 3 or 14 d after the last injection. For AMPA receptor subunits,
quantitative in situ hybridization studies showed no changes in GluR1-3 mRNA
levels in the NAc after 3 d, but decreases in GluR1 and GluR2 mRNA levels
were observed after 14 d (41). Parallel changes were observed at the protein

level using quantative immunoautoradiography (42). Similarly, mRNA and
protein levels for NR1 in the NAc were not altered by repeated amphetamine
at the 3-d withdrawal time, but both were signifi cantly decreased after 14 d of
withdrawal (24). The decreased levels of GluR1, GluR2, and NR1 subunits in
amphetamine-treated rats may be functionally signifi cant. Single-unit recording
studies performed in the NAc of rats treated with the same amphetamine
regimen, or a sensitizing regimen of cocaine, revealed that NAc neurons
recorded from drug-treated rats were subsensitive to glutamate as compared
to NAc neurons from saline-pretreated rats (9). Follow-up studies showed
that NAc neurons were also subsensitive to NMDA and AMPA but not a
metabotropic glutamate receptor agonist (Hu and White, unpublished observa-
tions). However, the correspondence is not perfect. The decreases in glutamate
receptor subunit expression were observed only after 14 d of withdrawal,
whereas electrophysiological subsensitivity was observed after both 3 and 14
days of withdrawal. Perhaps other mechanisms account for subsensitivity at
the early withdrawal time (see ref. 43). Another problem is that NAc neurons
recorded from repeated cocaine treated rats also show electrophysiological
subsensitivity to glutamate agonists (see previous discussion in this subhead-
ing), but most studies report increased glutamate receptor expression after
long withdrawals from repeated cocaine administration (see following portions
of this subheading).
Similar to our results showing no changes in glutamate receptor subunit
expression in the NAc 3 d after discontinuing repeated amphetamine, Fitzgerald
et al. (12) found no change in NAc levels of GluR1, GluR2, NR1, NR2A/B,
GluR6/7, and KA-2 subunit proteins (measured by Western blotting) 16–18 h
20 Wolf
after withdrawal from repeated cocaine treatment. However, alterations are
observed at later withdrawal times, and they differ from those produced by
amphetamine. Churchill et al. (14) used Western blotting to determine protein
levels of glutamate receptor subunits 24 h or 3 wk after discontinuing daily

cocaine or saline injections (see Subheading 2.2. for more details). After 24 h,
there were no changes in GluR1 or NMDAR1 levels in the NAc, consistent
with the fi ndings of Fitzgerald et al. (12). However, after 3 wk, sensitized
rats (but not cocaine-treated rats that failed to sensitize) showed a signifi cant
increase in GluR1 levels in the NAc compared to saline-treated rats. When
saline-treated rats were compared to all cocaine rats (sensitized + nonsensi-
tized), there was a trend toward increased NMDAR1 in the NAc after repeated
cocaine, but this was actually more pronounced in nonsensitized rats. GluR2/3
was not changed in the NAc at either withdrawal time. Dorsal striatum was
analyzed only after 3 wk of withdrawal; there were no changes in GluR1,
GluR2/3, or NR1. Likewise, these subunits were unchanged in prefrontal
cortex or VTA after 3 wk of withdrawal, although increases in GluR1 and
NR1 were found in VTA of sensitized rats 24 h after discontinuing cocaine
(see Subheading 2.2.).
Interestingly, the changes in protein levels found by Churchill et al. (14)
were not paralleled by changes at the mRNA level. Ghasemzadeh et al. (22)
used in situ hybridization histochemistry and RT-PCR to quantify glutamate
receptor subunit mRNA levels 3 wk after discontinuing the same regimen of
cocaine or saline injections used by Churchill et al. (14). Twenty-four hours
before decapitation, half the rats in each group were challenged with saline and
half with cocaine. In NAc, acute cocaine decreased mRNA levels for GluR3,
GluR4, and NR1, while repeated cocaine also decreased GluR3 mRNA and
increased mGluR5 mRNA. The only signifi cant effect in dorsolateral striatum
was decreased NR1 mRNA after acute cocaine. The VTA and PFC were also
evaluated (see Subheadings 2.2. and 4.2.). Because of the complexity of
the design, the reader should consult the article for an in-depth discussion
of interactions between chronic cocaine treatment and acute challenge, and
interesting trends that were apparent in some groups.
Scheggi et al. (44) used Western blotting to measure glutamate receptor
subunits after administering 40 mg/kg of cocaine every other day over 14 d,

testing for sensitization after 10 d of withdrawal, and killing the rats 1 wk after
the test for sensitization. In NAc, signifi cant increases in GluR1, NR1, and
NR2B (but not GluR2 or NR2A) were found in sensitized rats. The changes in
GluR1 and NR1 are in agreement with those reported by Churchill et al. (14). In
hippocampus, only the NR2B subunit was signifi cantly elevated although there
was a trend toward increased NR1 (26% increase). In the PFC, small increases
(~20%) were observed for NR1 and NR2B, but these were not signifi cant, and
Psychostimulants and Glutamate Receptors 21
there was no change in GluR1. All of these changes were blocked if MK-801
was continuously infused (s.c., via osmotic minipumps) during cocaine
administration, a treatment that also blocked development of sensitization,
suggesting they are linked to sensitization.
Chronic cocaine treatment leads to accumulation in some NAc neurons of
stable isoforms of the transcription factor ∆FosB, so Kelz et al. (45) used
transgenic mice in which ∆FosB was induced in a subset of NAc neurons to
model chronic cocaine treatment. These mice showed increased responsiveness
to rewarding and locomotor-activating effects of cocaine, as well as increased
expression of GluR2 in the NAc but not dorsal striatum. In a place conditioning
test, rats that received intra-NAc injections of a recombinant herpes simplex
virus vector encoding GluR2 spent more time in a cocaine-paired chamber
than controls, while rats made to overexpress GluR1 spent less time in the
cocaine-paired environment. Although this suggests that increased NAc levels
of GluR2 may account for enhanced rewarding effects of cocaine in the ∆FosB-
expressing mice, more work is needed to evaluate the relevance of these
fi ndings to the intact cocaine-treated animal.
NR2B is an interesting NMDAR subunit, as it is implicated in ethanol
dependence (46) and morphine-induced conditioned place preference (47).
Loftis and Janowsky (23) measured NR2B levels using immunohistochemical
methods in NAc and dorsolateral neostriatum, as well as hippocampal and
cortical regions (see Subheading 4.2.). Rats were treated with 20 mg/kg of

cocaine × 7 d (or saline) and killed 24 h, 72 h, or 14 d after discontinuing
injections. In dorsal striatum, there were no changes after 24 or 72 h, but NR2B
immunolabeling was increased after 14 d. In the NAc, NR2B was decreased
in shell but not core after 24 h, no changes were present after 72 h, and there
were increases in core and shell after 14 d.
Several recent studies have evaluated glutamate receptor binding after
repeated cocaine. Keys and Ellison (48) found a decrease in [
3
H]AMPA
binding, assessed with autoradiography, in ventral striatum, and a trend in NAc,
21 d following two exposures to cocaine administered continuously for 5 d
via subcutaneous pellets. Itzhak and Martin (49) compared NMDA receptor
binding in several brain regions (striatum, amygdala, and hippocampus) in
rats treated for 5 d with 15 mg/kg of cocaine (a sensitizing regimen) and mice
treated for the same time with a higher dose of cocaine (35 mg/kg; a regimen
that resulted in kindled seizures). No changes in NMDA receptor binding were
found with the sensitizing regimen, whereas binding was elevated in all regions
3 d after the high-dose regimen was discontinued, with additional alterations
occurring after the expression of kindled seizures. Szumlinski et al. (50) found
no changes in [
3
H]MK-801 binding in the rat striatum after a sensitizing
regimen of cocaine (fi ve daily injections of 15 mg/kg of cocaine) and 2 wk
22 Wolf
of withdrawal. Bhargava and Kumar (51) treated mice with a sensitizing
regimen of cocaine (10 mg/kg, twice daily for 7 d). Immediately after drug
treatment, [
3
H]MK-801 binding was increased in cerebellum and spinal cord
but decreased in cortex and hypothalamus. After withdrawal, binding remained

decreased in cortex but other changes normalized.
Recent studies have focused on the role of metabotropic glutamate receptors
in sensitization. Mao and Wang (52) used quantitative in situ hybridization
histochemistry to measure mRNA levels for group I mGluRs (mGluR1 and
mGluR5) in the NAc and striatum in naïve and amphetamine-sensitized rats.
No changes in mGluR1 or mGluR5 mRNA levels were observed in naïve rats
3 h after acute administration of amphetamine. In contrast, 3 h after the last
of fi ve daily amphetamine injections, mGluR1 mRNA levels were increased
in dorsal striatum and NAc. This effect was transient, as no changes were
observed after 7, 14, or 28 d of withdrawal. A different pattern was observed
for mGluR5. Levels of mRNA were decreased markedly 3 h after the fi nal
amphetamine injection, and the reduction persisted at 7-, 14-, and 28-d
withdrawal times. In a rare example of concordance between amphetamine and
cocaine fi ndings, Swanson et al. (53) found a small but signifi cant reduction
in mGluR5 protein levels, measured by Western blotting, in the medial NAc of
rats killed 3 wk after discontinuation of repeated cocaine injections. mGluR5
is postsynaptic and can negatively modulate AMPA receptor transmission.
Thus, the authors suggested that cocaine-induced decreases in mGluR5 may
contribute to the potentiation of AMPA receptor-mediated behavioral responses
related to drug-seeking behavior that have been reported after chronic cocaine
administration (54,55). In the same study, repeated cocaine administration
attenuated the ability of mGluR1 stimulation to decrease glutamate release and
locomotor activity, but this was not accompanied by alterations in mGluR1
protein levels and may be attributable to altered expression of Homer1b/c, a
scaffolding protein that regulates mGluR signaling (53). Increasing evidence
indicates that mGluRs play an important role in behavioral responses to
psychomotor stimulants (56).
A relatively unexplored question, owing primarily to technical diffi culty, is
whether posttranslational modifi cation of glutamate receptors is altered after
repeated drug treatment. Bibb et al. (57) found reduced peak amplitudes of

AMPA/kainate-evoked currents in acutely dissociated striatal neurons from
rats chronically treated with cocaine; other fi ndings suggested that this was
attributable to reduced PKA-dependent phosphorylation of GluR1.
3.3. Summary: NAc and Striatum
As discussed in Subheading 3.1., considerable evidence implicates gluta-
mate receptors in the striatal complex in persistent neuroadaptations associ-
Psychostimulants and Glutamate Receptors 23
ated with behavioral sensitization and drug-seeking behavior. There is some
agreement that GluR1 and NR1 levels are not altered in the NAc after short
withdrawals (1–3 d) from repeated cocaine or amphetamine administration. At
longer withdrawal times (2–3 wk), cocaine-treated rats may show increases in
GluR1, NR1, and NR2B, whereas amphetamine-treated rats show decreases
in GluR1, GluR2, and NR1. There may also be persistent changes in the
expression and function of group I mGluRs. The delayed onset of many of the
reported changes in glutamate receptor expression is consistent with a role for
the NAc in the long-term maintenance of sensitization and other drug-induced
behavioral changes. However, it is diffi cult to reconcile opposite effects of
cocaine and amphetamine on glutamate receptor expression with a role for
these changes in the maintenance and expression of sensitization, as both
drugs produce similar behavioral effects (augmented locomotor response) in
sensitized rats. It should be kept in mind that the NAc contains heterogeneous
populations of projection neurons and interneurons, and we do not know the
phenotype of the neurons that experience changes in glutamate receptor subunit
expression (e.g., 42,52). Moreover, other types of drug-induced changes
may contribute importantly to the excitability of NAc neurons. For example,
Zhang et al. (43) found reduced sodium currents in NAc neurons after a short
withdrawal from repeated cocaine, while Thomas et al. (58) found evidence
for LTD in the NAc after long-term withdrawal from cocaine. In fact, growing
evidence suggests that abnormal synaptic plasticity in the NAc, triggered
by chronic drug treatment, leads to dysregulation of motivation- and reward-

related circuits and thereby contributes to addiction (2). It will be important to
determine whether alterations in glutamate receptor expression contribute to
the induction of altered plasticity, are involved in its expression, or represent
compensatory responses to changes in the activity of glutamate-containing
projections.
4. Effect of Psychomotor Stimulants on Glutamate Receptor
Expression in the PFC and Other Cortical or Limbic Regions
4.1. Role of the Prefrontal Cortex in Behavioral Sensitization
The PFC is now acknowledged to play an important role in behavioral
sensitization. Excitotoxic lesions of the PFC prevent the development of
sensitization (59–61) as well as cellular changes in DA systems that are
closely associated with sensitization (61). The role of PFC in the expression
of behavioral sensitization in response to psychostimulant challenge is more
controversial. Some evidence suggests that expression of sensitization requires
glutamatergic transmission between the dorsal PFC and the NAc core (62).
On the other hand, excitotoxic lesions of the PFC that are suffi cient to prevent
24 Wolf
development of sensitization do not interfere with expression (63,64). Other
fi ndings suggest that maintenance and expression of sensitization may be
associated with loss of inhibitory DA tone in the PFC, leading to a loss
of inhibitory control over PFC projections to subcortical regions; multiple
mechanisms may contribute (e.g., 65–69). Less has been done to examine
specifically the role of glutamate transmission in the PFC in behavioral
sensitization. No studies have examined the effect of intra-PFC injection of
glutamate receptor antagonists and only a few microdialysis studies have
assessed glutamate release in the PFC in response to stimulants (70–73).
Likewise, there have been relatively few studies on stimulant-induced altera-
tions in glutamate receptor expression in the PFC as compared to the striatum
and midbrain.
4.2. Results in the PFC

Using quantitative in situ hybridization and immunoautoradiography, we
found increased GluR1 mRNA and protein levels 3 d after discontinuing
repeated amphetamine administration; this effect was transient, as it was not
observed in rats killed after 14 d of withdrawal (41,42). This increase in GluR1
may be functionally signifi cant, as PFC neurons recorded from amphetamine-
treated rats after 3 d of withdrawal (but not 14 d of withdrawal) showed
increased responsiveness to the excitatory effects of iontophoretically applied
glutamate (67). In studies using the same amphetamine regimen, we found
a signifi cant decrease in NR1 mRNA levels and a trend toward decreased
immunolabeling after 14 d of withdrawal, but no change after 3 d (24).
Cocaine also exerts complex effects on glutamate receptor subunit expres-
sion in the PFC. Churchill et al. (14) found no changes in PFC levels of GluR1,
GluR2/3, or NMDAR1 (using Western blots) 3 wk after discontinuing a week
of daily cocaine injections (see Subheading 2.2. for more details on this
study). In another study, rats were treated with cocaine, tested for sensitization
after 10 d of withdrawal, and killed 1 wk after the test for sensitization (44;
see Subheading 3.2. for more details). Small increases (~20%) were observed
for NR1 and NR2B in the PFC, but these were not signifi cant, and there was
no change in GluR1. Loftis and Janowsky (23) measured NR2B levels using
immunohistochemical methods in VTA and NAc (see Subheadings 2.2. and
3.2.), as well as dorsolateral neostriatum, the hippocampal formation (CA1,
CA3, and dentate gyrus), and the cortex (medial frontal cortex, lateral frontal
cortex, and parietal cortex). Rats were killed 24 h, 72 h, or 14 d after discontinu-
ation of repeated cocaine or saline injections. There were no changes in
the hippocampal formation following 24 or 72 h of withdrawal. Results in
cortex depended on the region analyzed. For medial frontal cortex, there were
Psychostimulants and Glutamate Receptors 25
increases at all withdrawal times. For lateral frontal cortex and parietal cortex,
there was no change after 24 h, but increases after 72 h and 14 d. This study
also measured neuronal nitric oxide synthase, but these results will not be

discussed.
4.3. Summary: PFC and Other Cortical and Limbic Regions
Glutamate receptor expression in the PFC undergoes complex changes after
drug administration is discontinued that depend on the withdrawal time and
probably differ between cocaine and amphetamine, at least for NR1. In general,
some results suggest that AMPA receptor subunit expression changes at early
withdrawal times whereas NMDA receptor subunit expression is altered after
longer withdrawals. Because relatively few studies have assessed glutamate
transmission in the PFC of sensitized rats using electrophysiological or
neurochemical approaches, it is diffi cult to assess the functional signifi cance
of observed changes. An exception is the correlation between increased
responsiveness of PFC neurons to glutamate (67), and increased expression of
GluR1 in the PFC (41,42), after short withdrawals from repeated amphetamine
administration. It will be important to conduct studies on additional brain
regions implicated in addiction, such as the amygdala.
5. Conclusions
It is clear that repeated administration of cocaine or amphetamine infl u-
ences glutamate receptor expression in brain regions important for behavioral
sensitization and addiction. However, to date, the data obtained raise more
questions than they answer. One important problem is that amphetamine and
cocaine produce different patterns of changes, whereas both produce behavioral
sensitization. Either there are multiple ways to achieve a sensitized state, or
the changes in glutamate receptor expression are not directly associated with
sensitization. The picture is made more complex by different effects at different
withdrawal times, different effects with different drug regimens, and lack of
agreement between laboratories using similar drug regimens. Another problem
is that studies of receptor expression have been conducted at the regional
level, precluding identification of the types of cells exhibiting particular
alterations in glutamate receptor expression after stimulant exposure. Without
such information, it is hard to predict the functional effect of these alterations

at the level of neuronal circuits. For example, does the increase in GluR1
expression in the PFC after repeated amphetamine occur in pyramidal neurons
or interneurons, or in a subset of one of these populations? It will be important
to conduct future studies in identifi ed cells, although this is a very challenging
26 Wolf
undertaking. It will also be important to study the cellular mechanisms by which
monoamine-releasing psychomotor stimulants infl uence the expression of
glutamate receptors, as well as other aspects of glutamate neurotransmission.
References
1. Hyman, S. E. and Malenka, R. C. (2001) Addiction and the brain: the neurobiology
of compulsion and its persistence. Nat. Rev. 2, 695–703.
2. Wolf, M. E. (2002) Addiction and glutamate-dependent plasticity, in Glutamate
and Addiction (Herman, B. H., Frankenheim, J., Litten, R., Sheridan, P. H., Weight,
F. F., and Zukin, S. R., eds.), Humana Press, Totowa, NJ, pp. 127–142.
3. Jentsch, J. D. and Taylor, J. R. (1999) Impulsivity resulting from frontostriatal
dysfunction in drug abuse: implications for the control of behavior by reward-
related stimuli. Psychopharmacology 146, 373–390.
4. Everitt, B. J. and Wolf, M. E. (2002) Psychomotor stimulant addiction: a neural
systems perspective. J. Neurosci. 22, 3312–3320.
5. Wolf, M. E. (2001) The neuroplasticity of addiction, in Toward a Theory of
Neuroplasticity (Shaw, C. A. and McEachern, J. C., eds.), Taylor & Francis,
Philadelphia, pp. 359–372.
6. Robinson, T. E. and Berridge, K. C. (1993) The neural basis of drug craving: an
incentive-sensitization theory of addiction. Brain Res. Rev. 18, 247–291.
7. Robinson, T. E. and Berridge, K. C. (2000) The psychology and neurobiology of
addiction: an incentive-sensitization view. Addiction 95 (Suppl. 2), S91–S117.
8. Wolf, M. E. (1998) The role of excitatory amino acids in behavioral sensitization
to psychomotor stimulants. Prog. Neurobiol. 54, 679–720.
9. White, F. J., Hu, X T., Zhang, X F., and Wolf, M. E. (1995) Repeated administra-
tion of cocaine or amphetamine alters neuronal responses to glutamate in the

mesoaccumbens dopamine system. J. Pharmacol. Exp. Ther. 273, 445–454.
10. Zhang, X F., Hu, X T., White, F. J., and Wolf, M. E. (1997) Increased responsive-
ness of ventral tegmental area dopamine neurons to glutamate after repeated
administration of cocaine or amphetamine is transient and selectively involves
AMPA receptors. J. Pharmacol. Exp. Ther. 281, 699–706.
11. Giorgetti, M., Hotsenpiller, G., Ward, P., Teppen, T., and Wolf, M. E. (2001)
Amphetamine-induced plasticity of AMPA receptors in the ventral tegmental area:
effects on extracellular levels of dopamine and glutamate in freely moving rats.
J. Neurosci 21, 6362–6369.
12. Fitzgerald, L. W., Ortiz, J., Hamedani, A. G., and Nestler, E. J. (1996) Drugs
of abuse and stress increase the expression of GluR1 and NMDAR1 glutamate
receptor subunits in the rat ventral tegmental area: common adaptations among
cross-sensitizing agents. J. Neurosci. 16, 274–282.
13. Ortiz, J., Fitzgerald, L. W., Charlton, M., et al. (1995) Biochemical actions
of chronic ethanol exposure in the mesolimbic dopamine system. Synapse 21,
289–298.